Effects of follicle stimulating hormone (FSH) on follicular development, oocyte retrieval, and in vitro fertilization (IVF) in ewes during breeding season and seasonal anestrus

Administration of FSH increases the number of developing follicles, and affects oocyte health and cleavage rate. To determine the optimal level of FSH treatment, studies were conducted during the normal breeding season and seasonal anestrus. In Experiment 1, ewes were implanted with SyncroMate-B (SMB; norgestomet) for 14 days during the breeding season. Beginning on day 12 or 13 after SMB implantation, ewes were treated with saline (control; n=10), or treated with FSH for two days (2D; n=9) or three days (3D; n=10). In Experiment 2, conducted during seasonal anestrus, ewes were implanted with SMB for 14 days (n=23) or were not implanted (n=26). The SMB-implanted and nonimplanted ewes were assigned to one of three treatments as in Experiment 1: control (n=13), 2D (n=21) or 3D (n=15). In Experiments 1 and 2, ewes were laparotomized to count the number of follicles < or = 3 mm and > 3 mm and to retrieve oocytes. Healthy oocytes from each treatment were used for IVF. In Experiment 3, ewes (n=6) were implanted twice with SMB for 14 days during seasonal anestrus. Ewes were injected with FSH for 2 days, and the oocytes were collected and fertilized as in Experiments 1 and 2. In Experiment 1, FSH-treatment increased (P < 0.05) the number of follicles > 3 mm, the number of oocytes retrieved from follicles < or = 3 mm and > 3 mm, the proportion of healthy oocytes, and the number of oocytes used for IVF. Oocytes from control and 2D ewes had greater (P < 0.01) cleavage rates than 3D ewes (68% and 71% vs. 42%). In Experiment 2, implanted and nonimplanted ewes had similar (P > 0.05) numbers of follicles, total oocytes, and healthy oocytes; therefore, data were combined. The FSH treatment increased (P < 0.01) the number of follicles > 3 mm, and the number of oocytes recovered from follicles > 3 mm. The recovery rate of oocytes and the percentage of healthy oocytes were similar for control and FSH-treated ewes. The cleavage rate in Experiment 2 ranged from 4 to 16%. In Experiment 3, the cleavage rate for ewes treated twice with SMB was 27% which tended to be greater (P < 0.07) than for the 2D ewes that received one SMB implant in Experiment 2. These data indicate that FSH increased the number of developing follicles and the number of healthy oocytes retrieved from ewes during the breeding season and seasonal anestrus. However, cleavage rates during seasonal anestrus were lower than during the normal breeding season in both FSH-treated and control ewes. Treatment of ewes for 2 days with FSH resulted in a greater cleavage rate than treatment of ewes for 3 days.     

Short-term preservation of porcine oocytes in ambient temperature: novel approaches

The objective of this study was to evaluate the feasibility of preserving porcine oocytes without freezing. To optimize preservation conditions, porcine cumulus-oocyte complexes (COCs) were preserved in TCM-199, porcine follicular fluid (pFF) and FCS at different temperatures (4°C, 20°C, 25°C, 27.5°C, 30°C and 38.5°C) for 1 day, 2 days or 3 days. After preservation, oocyte morphology, germinal vesicle (GV) rate, actin cytoskeleton organization, cortical granule distribution, mitochondrial translocation and intracellular glutathione level were evaluated. Oocyte maturation was indicated by first polar body emission and spindle morphology after in vitro culture. Strikingly, when COCs were stored at 27.5°C for 3 days in pFF or FCS, more than 60% oocytes were still arrested at the GV stage and more than 50% oocytes matured into MII stages after culture. Almost 80% oocytes showed normal actin organization and cortical granule relocation to the cortex, and approximately 50% oocytes showed diffused mitochondria distribution patterns and normal spindle configurations. While stored in TCM-199, all these criteria decreased significantly. Glutathione (GSH) level in the pFF or FCS group was higher than in the TCM-199 group, but lower than in the non-preserved control group. The preserved oocytes could be fertilized and developed to blastocysts (about 10%) with normal cell number, which is clear evidence for their retaining the developmental potentiality after 3d preservation. Thus, we have developed a simple method for preserving immature pig oocytes at an ambient temperature for several days without evident damage of cytoplasm and keeping oocyte developmental competence.     

The heat-shock response in xenopus oocytes is controlled at the translational level

Xenopus laevis oocytes respond to high temperature (>31°C) by the synthesis of one major (70 kilodalton) protein and by a gradual reduction in the rate of normal protein synthesis. In contrast with most other cells, the heat-shock response of Xenopus oocytes is controlled exclusively at the translational level. Enucleated or α-amanitin-injected oocytes synthesize normal levels of heat-shock protein. Thus high temperature induces the translation of preformed heat-shock mRNA. This continues for more than a day after a shift back to a normal temperature, but ceases within 2 days. Heat-shock protein synthesis can be sequentially induced and inactivated in the same oocyte over several days. We conclude that an oocyte contains 10–100 pg of heat-shock mRNA, which is synthesized during oogenesis at the normal temperature, and which is stored in an inactive state by a “masking” mechanism.     

Effects of eCG and FSH on ovarian response, recovery rate and number and quality of oocytes obtained by ovum pick-up in Holstein cows

The goal of the present study was to compare the ovarian response, oocyte yields per animal, and the morphological quality of oocytes collected by ultrasound guided follicular aspiration from Holstein cows treated either with FSH or eCG. Twenty four normal cyclic, German Holstein cows were randomly divided into two groups. Fourteen cows received 3000 IU eCG on day-4 prior to ovum pick-up (OPU) (day 0), 2 days later (day-2), 625 microg cloprostenol was administered. On day-1 GnRH was administered i.m. and 24h later OPU (day 0) was performed. In ten cows a total dose of 500 IU follicle stimulating hormone (Pluset) was administered intramuscularly in a constant dosage for 4 days with intervals of 12h, starting on day-5. Luteolysis was induced by application of 625 microg cloprostenol on day-2. On day-1 (24h after the last FSH treatment) GnRH was administered i.m. and 24h later OPU (day 0) was performed. Ovarian follicles were visualized on the ultrasound monitor, counted and recorded. All visible antral follicles were punctured. Recovered oocytes were graded morphologically based on the cumulus investment. Average follicle number in ovaries was higher in FSH group than eCG group (p<0.05). Oocyte yields per animal did not differ between FSH and eCG groups. The proportion of grade A oocytes was higher in the FSH group in the than eCG group (p<0.05). Likewise, rate of grade C oocytes in FSH group were lower than eCG group (p<0.05). In conclusion, these results suggest that ovarian response, follicle number in ovaries and oocyte quality are affected by the type of gonadotropin and FSH is better alternative than eCG for OPU treatment.     

Leptin accelerates pronuclear formation following intracytoplasmic sperm injection of porcine oocytes: Possible role for MAP kinase inactivation

Leptin, a multifunctional hormone, is present in mammalian oocytes and follicular fluids and cumulus cells. While leptin modulates oocyte maturation in vitro which seems to result in enhancement of embryo development, it is unclear whether leptin treatment of oocytes affects cytoplasmic maturation and fertilization processes. In order to gain a better understanding of the role of leptin during oocyte maturation, we examined microtubule and microfilament assembly following oocyte maturation and blastocyst formation, mitogen-activated protein kinase (MAPK) activity, and pronuclear formation following parthenogenetic stimuli or intracytoplasmic sperm injection (ICSI) in leptin-treated oocytes. Addition of 10 or 100 ng/ml leptin during oocyte maturation did not increase the proportion of metaphase II oocytes, but enhanced development to blastocyst stage by day 7 (P < 0.01) after parthenogenetic activation (PA), accompanied by increased cell number. However there was no effect on the number of apoptotic cells in blastocysts. Following maturation in the presence of leptin, there were more oocytes with normal spindle formation. MAPK activity decreased more rapidly, and pronuclear formation was accelerated after parthenogenetic activation or ICSI of leptin-treated oocytes. These results suggested that exogeneous leptin enhanced spindle assembly and accelerated pronuclear formation following fertilization, possibly via the MAPK pathway.     

Activation of protein kinase C suppresses fragmentation of pig oocytes aged in vitro

When cultured for an extended time, pig oocytes that matured in vitro to the stage of metaphase II undergo the complex process designated as ageing. Under our conditions, some pig oocytes aged 3 days remained at the stage of metaphase II (22%), but others underwent spontaneous parthenogenetic activation (45%), and still others perished through fragmentation (28%) or lysis (5%). Activation of protein kinases C (PKCs) using phorbol-12-myristate-13-acetate (PMA) protects oocytes from fragmentation. None of the oocytes were fragmented after 3 days of aging in 50 nM of PMA. A similar effect (8% of fragmented oocytes) was observed after a 3-day treatment of aging oocytes with 100 μM of 1-stearoyl-2arachidonoyl-sn-glycerol (STEAR). PMA and STEAR activate both calcium-dependent and calcium-independent PKCs. This combined effect on PKCs seems to be essential for the protection of oocytes from fragmentation. Neither the specific activator of calcium-dependent PKCs 1-oleoyl-2-acetyl-sn-glycerol (OLE) nor the specific activator of calcium-independent PKCs dipalmitoyl-l-α-phosphatidylinositol-3,4,5-triphosphate heptaammonium salt (DIPALM) suppressed the fragmentation of aging pig oocytes. Twenty-one percentage of oocytes fragmented when aged for 3 days in 10 μM OLE and 26% of aged oocytes fragmented in 100 nM of DIPALM. However, fragmentation was significantly suppressed to 7% when the oocytes were exposed to the combination of both 10 μM OLE and 100 nM DIPALM. Aging pig oocytes cultured for 1 day with PMA maintained a high capability of being parthenogenetically activated (86% of activated oocytes), using calcium ionophore with 6-dimethylaminopurine. Ageing oocytes treated with PMA also had high capability of cleavage (82%) after their artificial parthenogenetic activation. However, their ability to develop to the stage of blastocyst (12%) was suppressed when compared with oocytes activated immediately after their maturation (29%).     

饥饿小鼠卵母细胞中自噬和凋亡的电镜研究

小鼠(Mus musculus domesticus)原始卵泡形成在出生后3 d内进行得最剧烈,此时有大量卵母细胞丢失。出生后不久原始卵泡库就建立起来,新生鼠都会经历一段时间饥饿再摄入母乳营养,对出生后的子鼠饥饿处理时,出现了自噬和凋亡的动态变化,自噬和凋亡都可以影响细胞的存活,这很可能与卵母细胞的大量丢失有关。在本项研究中,将对照组子鼠正常母乳喂养,处理组子鼠与母鼠分开,完全不给予母乳。分别收取饥饿1.5 d与2 d子鼠的卵巢制作电镜切片,每组3只子鼠,每只子鼠3张电镜切片,每组共统计9张切片。在电镜下观察其形态变化。通过观察发现,饥饿1.5 d的子鼠卵巢与正常1.5d的子鼠卵巢相比,卵母细胞中的自噬小体数量显著增加。这表明,饥饿处理1.5d促进了卵母细胞的自噬,这可能有助于维持卵母细胞的形态及存活。饥饿处理2 d的子鼠卵巢显示出不同的结果。饥饿2 d的子鼠处于生命的临界阶段,已出现小部分个体死亡。存活子鼠卵巢的电镜形态学观察发现,与正常哺乳2 d的子鼠卵母细胞相比,饥饿2 d子鼠卵母细胞中自噬小体的数量显著减少,并出现了多数卵母细胞凋亡的现象,出现许多凋亡小体。本实验研究结果显示,...     

Anti-hyaluronidase oligosaccharide derived from chondroitin sulfate a effectively reduces polyspermy during in vitro fertilization of porcine oocytes

The present study was conducted to examine the effects of chondroitin sulfate A-derived oligosaccharide (ChSAO) on hyaluronidase activity and in vitro fertilization (IVF) parameters. The activity of hyaluronidase extracted from preincubated boar sperm was completely blocked by ChSAO at concentrations of 10 microg/ml or higher. After in vitro maturation of porcine cumulus-oocyte complexes, some oocytes were freed from their cumulus cells, and cumulus-intact or cumulus-free oocytes were inseminated with sperm in IVF medium containing various concentrations of ChSAO (0.1-100 microg/ml). In cumulus-intact oocytes, the penetration and the polyspermy rates (39% and 28%, respectively) were significantly decreased by treatment with 100 microg/ml ChSAO compared with those of oocytes treated without ChSAO (63% and 52%, respectively). On the contrary, in cumulus-free oocytes, the addition of 10-100 microg/ml ChSAO significantly reduced the polyspermy rate compared with the control (25-30% versus 53%, respectively), whereas ChSAO had no effect on sperm penetration. Interestingly, ChSAO added to IVF medium significantly decreased the number of sperm bound to the zona pellucida (ZP) of cumulus-free oocytes in a concentration-dependent manner between 0.1 and 100 microg/ml. However, ChSAO had no effect on the time course change in ZP modification after oocyte activation by electrostimulation and the incidence of the acrosome-reacted sperm. Treatment with 100 microg/ml ChSAO during IVF of cumulus-free oocytes significantly increased the proportion of development to the blastocyst stage after in vitro insemination. Therefore, the present findings indicate that hyaluronidase-inhibitory ChSAO is an efficient probe for promoting normal fertilization process in terms of an effective decrease in the incidence of polyspermy during IVF of porcine oocytes.     

Effect of ovarian superstimulation on COC collection and maturation in alpacas

The objective of the present study was to compare the ovarian follicular response, cumulus-oocyte complex (COC) collection rate, and maturational status of COC collected from alpacas subsequent to treatment with two different superstimulatory protocols. Alpacas (n=7 per group) were treated with: (1) 200mg of FSH im divided bid for 3d, plus a single i.v. dose of 1000IU hCG 24h after the last FSH treatment, or (2) 1200IU of eCG as a single i.m. dose, plus a single i.v. dose of 1000IU of hCG on day 3 after eCG treatment (day 0=start of superstimulatory treatment). At 20-24h post-hCG treatment, the ovaries were surgically exposed and COC were collected by needle aspiration of all follicles > or =6mm. The FSH and eCG treatment groups did not differ with respect to the number of follicles > or =6mm at the time of COC collection (20.0+/-7.5 versus 27.0+/-3.3; P=0.5), the number of COC collected (26.2+/-8.4 versus 23.3+/-3.7; P=0.7), or the collection rate per follicle aspirated (89% versus 87%; P=0.7). No differences were detected between FSH- and eCG-treated alpacas in the number of expanded COC collected per alpaca (11.5+/-2.9 versus 8.8+/-2.8; P=0.54), the number of expanded COC in metaphase II (8.5+/-1.9 versus 6.0+/-2.1; P=0.1), or the number of compact COC with > or =3 layers of cumulus cells (12.5+/-4.3 versus 14.3+/-2.6; P=0.72). A greater proportion (P<0.05) of compact COC collected after FSH treatment matured in vitro to the metaphase II stage than after eCG treatment. Eight expanded alpaca COC were fertilized in vitro with llama sperm, three of which were fixed and stained 18h after exposure to sperm and five were cultured in vitro. Two of the three stained oocytes were in the pronuclear stage, and all five of the cultured oocytes developed to the two-cell and morula stages at 2 and 7 days, respectively, after in vitro fertilization. In summary, FSH and eCG treatments were equally effective for ovarian superstimulation and oocyte collection. Cumulus-oocyte complexes were collected from more than 80% of follicles aspirated during laparotomy. Nearly one third of the COC collected after superstimulation were in metaphase II, and more than 70% of the remaining COC progressed to metaphase II after in vitro maturation for 26h, bringing the mean number of oocytes available for in vitro fertilization to 16 per alpaca. Preliminary results support the hypothesis that alpaca oocytes obtained after superstimulation in the absence of progesterone are developmentally competent since morulae developed from all five COC fertilized and cultured in vitro.     

Localization of Period 1 mRNA in the ruminant oocyte and investigations of its role in ovarian function

The clock gene Period 1 (Per1) may be a prolificacy gene, because it localized to the mouse oocyte and Per1-null drosophila shed fewer eggs. Because Per1 mapped to a region of mouse chromosome 11 syntenic to bovine chromosome 19 where a quantitative trait loci (QTL) for ovulation rate existed, we hypothesized that Per1 influenced folliculogenesis and ovulation rate in ruminants. Ovarian cortex was collected at slaughter on days 5, 12, 15, 17, and 20 after estrus for real-time RT-PCR evaluation of Per1 mRNA expression in Dorset (n = 18), Romanov (n = 10), Romanov/Dorset (n = 21), and Composite (n = 22) ewes. Ovarian cortex was also collected from cows selected for increased ovulation rate (n=37) or unselected controls (n = 28) on days 4, 5, and 6 of the estrous cycle for in situ hybridization and real-time RT-PCR. To examine the role of Per1 in early follicular development, ovarian cortex from neonatal calves (n = 5) was cultured for 10 days and Per1 mRNA levels were measured on day 0 and on day 10 of culture. The primers generated a 483bp amplicon with 100% sequence homology to bovine RIGUI-like protein (Per1). In silico mapping of this sequence placed Per1 on bovine chromosome 19; however, it was 20cM from the QTL. Per1 mRNA expression was unaffected by prolificacy, day of the cycle, or pregnancy status in ewes or cows. The riboprobe hybridized to oocytes of bovine preantral and antral follicles. In bovine ovarian cortical cultures on day 0, the tissue contained mostly primordial follicles (5.6+/-0.6 follicles/section); however, after 10 days in culture, the number of primordial follicles per section decreased (0.5 follicles/section) and the number of primary follicles increased as follicles activated (day 0 = 0.5+/- 0.6 versus day 10 = 10.4 +/-0.6 primary follicles/section; P < 0.001). Per1 mRNA did not change over time in culture. We conclude that Per1 mRNA is expressed by ruminant oocytes in preantral and antral follicles; however, its physiological role in mammalian ovarian function remains to be elucidated.     

Effect of repeated eCG treatments and ovum pick-up on ovarian response and oocyte recovery during early pregnancy in suckling beef cows

This study was designed to evaluate in suckling early pregnant beef cows with and without eCG-pre-stimulation: (i) the influence of day gestation (from 40 to 101 days) and the consecutive eCG treatments on the follicular growth induced by means of ultrasound-guided transvaginal follicle ablation (FA; all follicles ≥ 5 mm) and the number and quality oocytes recovered by ovum pick-up (OPU) and (ii) the possible effects of repeated hormonal stimulation and FA/OPU on pregnancy outcome. Twelve suckling early pregnant Angus cows (40 days post fixed-time artificial insemination) were randomly assigned to each of two groups (n=6 group(-1)). Group 1 treatments included: FA (Day 0), eCG (1600 IU; Day 1) and OPU (Day 5). Group 2: as cited Group 1 with no eCG treatment. In both groups, OPU was repeated five times (Days 45, 59, 73, 87 and 101 of gestation). The numbers (mean ± SEM) of class II (5-9 mm; 4.3 ± 0.9) and class III (≥10 mm; 2.5 ± 0.4) follicles visualized per cow per OPU session in eCG-treated cows were greater (P<0.05) than for non-treated cows (0.9 ± 0.1 and 0.9 ± 0.1, respectively). In contrast, the number (mean ± SEM) of class I (<5mm) follicles per cow per OPU session was lower for cows with eCG treatment (2.8 ± 0.4) than for non-treated cows (5.7 ± 0.5). The mean number of aspirated follicles was not significantly different (P<0.05) between eCG-treated cows and non-treated cows at 45 and 59 days of pregnancy. However, the mean number of aspirated follicles was greater (P=0.03) in eCG-treated cows than non-treated cows from 73 day of pregnancy onwards. The numbers (mean ± SEM) of recovered oocytes and viable oocytes/cow/session were greater (P<0.05) for eCG-treated cows (2.2 ± 0.2 and 1.6 ± 0.4, respectively) than for non-treated cows (1.0 ± 0.2 and 0.9 ± 0.2, respectively). No donor pregnancies were lost either during or following OPU procedure. We can conclude that (1) eCG-treated pregnant suckled cows can be a source of oocytes for IVF at least to 100 days of gestation and (2) repeated FA/eCG treatment/OPU procedures did not affect the pregnancy outcome.     

Protein consumption by mated, unmated, sterile and fertile adults of the Queensland fruit fly, Bactrocera tryoni and its relation to egg production

Abstract. Female adults of Bactrocera tryoni (Froggatt) (Diptera: Tephritidae) at 25 °C require more than 0.1 mg of yeast autolysate per day to mature their oocytes to the vitellogenic stage and mate. Those given 0.2 mg per day from day 2 of adult life mated (when given the opportunity between 11 and 13 days) and each laid approximately 100 eggs (just over one egg per ovariole) by day 56. Females allowed to feed ad libitum from day 2, then 0 or 0.2 mg per day from day 14, laid approximately 75 and 100 eggs, respectively (after mating), whereas those fed ad libitum from day 2 to day 56 laid approximately 540 eggs after mating (averaging just over six eggs per ovariole). The developmental pattern of intake of normal females when on an ad libitum diet showed a rise to a peak at 5–7 days, followed by a decline to sustained low levels if not mated, but rising to a lower peak if mated between days 11–13 followed by a steady decline. Female flies that had been sterilized by 80 Gy gamma irradiation at the puparial stage had a pattern of food consumption similar to that of normal females mated at 7 days but they produced no yolky oocytes and had a darkened fat body. Normal and irradiated males had a feeding pattern similar to that of unmated nonirradiated females but at a lower level. The results are discussed in terms of the control of protein intake and the rate of its conversion to yolk.     

Ability to activate oocytes and chromosome integrity of mouse spermatozoa preserved in EGTA Tris-HCl buffered solution supplemented with antioxidants

Potential methods for cryopreservation of mouse spermatozoa are freeze-drying, desiccation, and suspension in EGTA Tris-HCl buffered solution (ETBS: 50 mM NaCl, 50 mM EGTA, and 10 mM Tris-HCl). To determine the duration that mouse spermatozoa suspended in ETBS-based solutions could retain their normal characteristics without freezing, spermatozoa collected from the cauda epididymis were suspended in ETBS or in ETBS supplemented with the antioxidants, dimethyl sulfoxide (DMSO), or DL-alpha-tocopherol acetate (Vitamin E acetate; VEA) diluted in DMSO, then held at ambient temperature (22-24 degrees C) for up to 9 days. When oocytes were injected with spermatozoa preserved in ETBS alone, activation rates of oocytes and chromosome integrity at the first cleavage metaphase decreased at 1 day (P < 0.001) and 2-4 days (P < 0.01) following treatment. When oocytes were injected with spermatozoa preserved in ETBS supplemented with DMSO or VEA/DMSO, chromosome integrity did not decrease significantly (through 9 days of preservation). Although DMSO maintained sperm chromosome integrity more effectively than VEA/DMSO up to 2-4 days (91 and 67%, normal karyotypes in DMSO and VEA/DMSO, respectively), VEA/DMSO helped to maintain the ability of spermatozoa to activate oocytes, but did not enhance the maintenance of sperm chromosome integrity. These results suggested that deterioration of spermatozoa preserved in ETBS alone was delayed by supplementation with antioxidants.     

Mitochondrial aggregation patterns and activity in in vitro cultured bovine oocytes recovered from early antral ovarian follicles

The low developmental competence seen in in vitro cultured oocytes collected from early antral follicles may be related to their mitochondrial status. The aim of this study was to examine the chromatin configuration, pattern of mitochondrial aggregation and mitochondrial activity of non-cultured and in vitro-cultured bovine oocytes originating from early antral ovarian follicles. Cumulus-oocyte complexes with adjacent granulosa cells (COCGs) were recovered from early antral follicles of 0.4 to 0.8 mm diameter. Control (Day 0) oocytes were recovered from freshly collected COCGs and fixed and stained. Selected COCGs were placed in growth culture for 7 days (Day 7) or 14 days (Day 14). Following growth culture, COCs with normal appearance were placed in maturation medium (IVM) for 24 h and then fixed and stained with MitoTracker CMTM Ros Orange and Hoechst 33258. The percentage of oocytes with an immature meiotic configuration after growth culture decreased with the time of growth culture, being 96.7; 72.5 and 35.4% respectively for Day 0, Day 7 and Day 14 of culture; the remaining oocytes were degenerating or resuming meiosis. After subsequent IVM the highest proportion of oocytes in diakinesis or metaphase I was found in the D7+IVM group (59.4%). When growth culture was prolonged to day 14 and IVM, the number of degenerated oocytes increased dramatically after IVM. The mitochondrial distribution in the oocytes changed from homogeneous to heterogeneous as growth culture time increased. The respiratory activity as measured by fluorescence intensity increased over the time of growth culture, and was highest in oocytes that had resumed GVBD. In conclusion, for oocytes in isolated COCGs from early antral follicles, culture conditions longer than 7 days should be more adapted for a slow nuclear maturation accompanied by a decreased energy metabolism to prevent chromatin pycnosis.     

In vitro embryo production in camel (Camelus dromedarius) from in vitro matured oocytes fertilized with epididymal spermatozoa stored at 4 degrees C

Experiments were conducted to study the effect of storing epididymal spermatozoa, in tris-tes- and tris-lactose egg yolk extenders, on their fertilizing ability and subsequent in vitro embryo development. Ovaries and testes were collected from a local slaughterhouse in normal saline solution (NSS) at 37 degrees C and on ice (0-1 degrees C), respectively. Cumulus oocyte complexes (COCs) aspirated from the follicles were randomly distributed to 4-well culture plates (20-25COCs/well) containing 500 microL of maturation medium and cultured at 38.5 degrees C in an atmosphere of 5% CO(2) in air for 36 h. Spermatozoa were collected from the cauda epididymides in syringes containing 2-3 mL of either tris-tes- or tris-lactose egg yolk extender. They were cooled down slowly and stored at refrigeration (4 degrees C) temperature. The spermatozoa were evaluated for motility and used for IVF of IVM oocytes on the day of collection and after 2, 4, 6 and 8 days of storage. On the day of IVF, spermatozoa were prepared by the swim up technique and both spermatozoa and oocytes were co-incubated at 38.5 degrees C in a humidified atmosphere of 5% CO(2) in air for 15-16 h. Presumptive zygotes were either fixed and stained with Hoechst 33342 for evaluation of fertilization or were cultured in 500 microL of the culture medium at 38.5 degrees C in an atmosphere of 5% CO(2), 5% O(2) and 90% N(2) in air. There was no significant difference (P>0.05) in the proportion of oocytes fertilized with spermatozoa stored in either of the two extenders for up to 8 days. The proportion of oocytes that cleaved (43-60%) and those that developed to blastocysts (14-21%) did not show any difference (P>0.05) either, when spermatozoa from different days of storage were used. First cleavage was observed as early as 16 h after IVF, early blastocysts had developed by day 4, expanded blastocysts after day 5 and hatching of blastocysts started after day 6 of culture. It may be concluded that dromedary epididymal spermatozoa survive in storage for at least 8 days in tris-lactose- and tris-tes egg yolk diluents at 4 degrees C. These spermatozoa maintain fertilizing ability and may be suitable for use in IVF and other assisted reproductive procedures.     

Developmental capability of rat oocytes matured in vitro in defined medium

The normality of in vitro matured oocytes was compared to that of in vivo matured (ovulated) oocytes at the following stages of development: germinal-vesicle breakdown, first polar body formation, fertilization (two polar bodies and two pronuclei with a sperm tail or first cleavage), and fetal development (day 20 fetuses). At all points, the in vitro oocytes exhibited a reduced ability, with oocytes matured cumulus-free having the poorest. The exposure of oocytes to human chorionic gonadotropin (hCG) for 2 hr before collection or during incubation improved their rates of maturation and development to day 20 fetuses but not their ability to undergo fertilization. While beneficial, the exposure to gonadotropins before or during maturation was not essential, as evidenced by the production of two day 20 fetuses matured and fertilized in vitro without any gonadotropin (luteinizing hormone or hCG) treatment in vivo or in vitro. These data demonstrate that in the population of in vitro matured oocytes there exist individuals wholly competent of complete normal development, albeit in a reduced proportion in comparison to normally matured and ovulated oocytes. That the in vitro handling, treatment, and culture of the oocytes may be responsible for some of the reduced developmental ability observed is suggested by the developmental abilities of ovulated oocytes under different conditions. Ovulated oocytes fertilized in the donor had the highest rates of development (46%), followed by those fertilized after transfer into mated recipients' oviducts (20%). The lowest rate was achieved with in vitro fertilized oocytes (7%), which represented the group subject to the greatest degree of manipulation and distinction from the normal in vivo process.     

Reversible inhibition of testicular androgen secretion by 3-, 5- and 6-month controlled-release microsphere formulations of the LH-RH agonist [D-Trp6, des-Gly-NH10(2)] LH-RH ethylamide in the dog

This study examines the effect of treatment with controlled-release poly(DL-lactide-coglycolide) microsphere formulations of the LH-RH agonist [D-Trp6, des-Gly-NH10(2)]-LH-RH ethylamide (LH-RH-A) designed to release about 100 or 200 micrograms of the peptide per day for 3, 5 or 6 months in male dogs. Plasma levels of testosterone and LH-RH-A were measured at 2-day intervals. After the first injection of the 100-micrograms/day formulation, plasma testosterone increased from 1.6 +/- 0.2 to 3.5 +/- 0.6 ng/ml for 5-7 days before decreasing and remaining at 0.05 +/- 0.008 ng/ml for approximately 150 days (5 months). After two months of recovery, microspheres designed to release 100 micrograms for 6 months of LH-RH agonist per day were then injected. Plasma testosterone levels showed an elevation from 1.5 +/- 0.5 to 4.7 +/- 2.0 ng/ml during the first few days before gradually decreasing to castration levels for 200 days (6 months). One month later, plasma testosterone had returned to normal levels. When microspheres designed to deliver an average of 200 micrograms per day of the peptide for 3 months were injected in another series of animals, castration levels of plasma testosterone were maintained for 95 days with a progressive increase to normal values at later time intervals. The animals of the first series of experiments were then sacrificed after 4 months of recovery following maintenance of plasma testosterone at castration levels for a total period of 11 months. The testes, prostate and pituitary gland were kept for histological examination which was completely normal in all tissues. The efficacy and excellent tolerance of the controlled-release form of LH-RH-A as inhibitor of the pituitary-gonadal axis strongly support the use of such long-term controlled-release formulations of LH-RH agonists for the treatment of sex steroid sensitive diseases.     

The influence of pre- and post-ovulatory insemination on sperm distribution in the oviduct,accessory sperm to the zona pellucida,fertilisation rate and embryo development in sows

The aim of present study was to investigate the influence of pre-compared with post-ovulatory insemination, on the distribution of spermatozoa in the oviduct, the accessory sperm counts on the zona pellucida and early embryonic development. Thirty-six crossbred multiparous sows (Swedish Landrace x Swedish Yorkshire) were artificially inseminated once either at 20-15 h before (group AIB) or at 15-20 h after (group AIA) ovulation by using a pooled semen of two boars. Thereafter, they were randomly allocated to one of five groups: slaughter at 5-6h after AI (group I-AIB), at 20-25 h after ovulation (groups II-AIB and II-AIA), at 70 h after ovulation (groups III-AIB and III-AIA), on day 11 (groups IV-AIB and IV-AIA, first day of standing oestrus=day 1) and on day 19 (groups V-AIB and V-AIA).The plasma levels of oestradiol-17beta and progesterone differed significantly (P相似文献   

Expression of heat shock protein70 in pig oocytes: heat shock response during oocyte growth

The heat shock response of growing and fully-grown pig oocytes was analyzed in vitro by determining heat shock protein70 (HSP70) synthesis under both normal conditions (39 degrees C; 0 and 6h) and after heat shock (43 degrees C; 1, 4 and 6h). The expression of HSP70 in oocytes was detected by immunoblotting analysis. Growing oocytes measuring 80-99 microm synthesized a high number of HSP70 without heat shock effect, and these were capable of increasing the synthesis of HSP70 after heat shock to a maximum after 1h. Growing oocytes measuring 100-115 microm also synthesized HSP70 without heat shock and after it, but the HSP70 synthesis was not statistically changed by increasing duration of heat shock. In fully-grown oocytes, great amounts of HSP70 were found without heat shock treatment, and the contents of HSP70 significantly decreased after heat shock. These results indicate that growing oocytes are able to synthesize HSP70 after heat shock. This ability declines at the end of the growth period, and fully-grown oocytes are unable to induce HSP70 synthesis after heat shock. HSP70 is synthesized and stored during oocyte growth. The high HSP70 synthesis in non-heat-treated growing oocytes and a great amount of HSP70 in fully-grown oocytes support the hypothesis that HSP70 is important for oocyte growth and maturation.     

In vitro maturation of bovine oocytes in the presence of growth hormone accelerates nuclear maturation and promotes subsequent embryonic development

Regulatory effect of GH on follicular growth and development in the cow is well documented. The aim of this study was to investigate the role of GH on in vitro bovine oocyte maturation. Therefore bovine cumulus oocyte complexes (COCs) were cultured in M199 without FCS and gonadotropins and in the presence of 10, 100, or 1,000 ng/ml bovine GH (NIH-GH-B18). The COCs were incubated at 39°C in a humidified atmosphere with 5% CO₂ in air and nuclear stage was assessed after 2, 4, 8, 16, 22, and 24 hr of incubation using DAPI staining. To assess the effect of GH on developmental capacity of the oocytes, COCs were incubated in the presence of GH for 22 hr, followed by IVF and in vitro embryo culture. Cultures without GH served as controls. For subsequent development, the embryos were cultured in M199 supplemented with 10% FCS on a monolayer of BRL cells. Embryos were scored morphologically and the efficiency of the culture system was evaluated as (1) the percentage of cleaved embryos 4 days after IVF, (2) the percentage of blastocysts on day 9 expressed on the basis of the number of oocytes at the onset of culture, and (3) the percentage of hatched blastocysts on day 11 expressed on the basis of the total number of blastocysts present at day 9. GH (100 and 1,000 ng/ml) significantly accelerated nuclear maturation (P < 0.001). A 4 and 8 h the percentage of oocytes in GV stage after GH treatment (54% and 19%) was significantly lower than the control (64% and 41%). Similarly at 16 and 22 h the percentage of oocytes in MII stage was significantly higher in the GH-treated group; (58% and 77%) and (46% and 62%) for GH and control respectively. The number of oocytes in MII beyond 22 hr of culture did not differ; 100 and 1,000 ng/ml GH induced significant cumulus expansion (P < 0.05), which was not observed in the absence of GH. Addition of 100 and 1,000 ng/ml GH during maturation significantly (P < 0.01) enhanced subsequent cleavage rate from (64% and 67%) in control to (75% and 81%) in GH-treated group; embryonic development in terms of day 9 blastocyst formation was also significantly increased in the presence of GH (29% and 34%) compared to the control (18% and 24%). The hatchability of the blastocysts was not influenced by GH. From the present data, it can be concluded that GH present during IVM has a beneficial effect on subsequent development. © 1996 Wiley-Liss, Inc.